KR101395344B1 - Peptide nucleic acids set for identifying raja kenojei mueller et henle species and identifying method of raja kenojei mueller et henle species using the same - Google Patents
Peptide nucleic acids set for identifying raja kenojei mueller et henle species and identifying method of raja kenojei mueller et henle species using the same Download PDFInfo
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Abstract
The present invention relates to a peptide nucleic acid set for identification of a hippocampus species and a method for distinguishing a hippocampus species using the same, and more particularly, to a method for discriminating a hippocampus species using one or more sequences comprising one or more base sequences selected from the group consisting of SEQ ID NOS: The present invention is characterized by hybridization of a peptide nucleic acid (PNA) with a sponge DNA sample to obtain a temperature-dependent melting curve while increasing the temperature of the hybridized product, and discriminating the spongy species from the melting temperature of the obtained melting curve. The present invention uses different PNAs having a strong binding force with DNA to exhibit different melting temperatures for different species of mackerel, so that the mackerel species can be identified easily, quickly, and accurately.
Description
The present invention relates to a peptide nucleic acid (PNA) set, a peptide nucleic acid kit, and a method for distinguishing a hippocampus using the same, and more particularly, to a method for identifying a hippocampal species using PNA So that it is possible to identify the different types of frying species in a simple, rapid and accurate manner.
The fish is a sea water belonging to the ray family. Its scientific name is Raja Kenojei MuLLER et HENLE. Body is rhombic and very wide. The head is small, the snout is protruding, the eyes are small, and the water hole is large. There is a small thorn on the center line of the back. Body light brown on back, white on white or gray. At the base of the pectoral fin is a large black door with a mottled pattern. The length of the body reaches about 150 cm. It is distributed in the coastal waters of Korea, the south coast of Japan, and the East China Sea. In Korea, there are many especially in the coastal waters of Busan, Mokpo, Yeonggwang and Incheon. It lives 20-80 meters deep. It is alive and spawning in spring.
These sponges are of high commercial value in Korea. Hongtung, which is cooked to taste with a rice wine, is most famous. In early spring, I put barley sprouts and red onion inside to boil spicy rice noodles, and eat them as sashimi, grilled, steamed and pung.
However, most of the domesticated sponges distributed in the country are distributed in a cutting state, and in terms of price, it is usual that domestic products are more expensive than foreign ones, and in some cases, foreign products are expensive, A technology that can easily and easily distinguish the origin or breed of the present invention is required.
Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and an object of the present invention is to provide a simple, rapid, and accurate identification of a sponge species using PNA having excellent binding force with DNA.
In addition, the present invention provides a peptide nucleic acid for discriminating a hippocampus species having a great difference in melting temperature (Tm) from a perfect match for perfect hybridization.
The present invention also relates to a peptide nucleic acid set for the determination of a haploid species, which is capable of detecting two or more nucleotide sequences with one peptide nucleic acid (or probe) and further comprising two or more peptide nucleic acids in one channel or tube , A kit, and a method for distinguishing a spider species using the kit.
In order to accomplish the above object, the present invention provides a peptide nucleic acid set for identifying a mare species comprising at least one peptide nucleic acid (PNA), wherein the peptide nucleic acid comprises at least one selected from the group consisting of SEQ ID NOS: 1 to 6 Or more of the above base sequences.
Here, it is preferable that the peptide nucleic acid has a reporter and a quencher at both ends thereof.
Each of the one or more base sequences is composed of a base in the range of 13 to 17, and may have a sequence corresponding to a single base polymorphism (SNP) region of the hippocampus at one or more of the 6th to 9th positions .
In another aspect of the present invention, there is provided a method for amplifying a protein comprising the steps of: hybridizing a proteinaceous sample with a peptide nucleic acid of the peptide nucleic acid set according to the present invention; Obtaining a temperature-dependent melting curve while increasing the temperature of the hybridized product; And determining a mare species from the melting temperature of the obtained melting curve.
Here, it is preferable that the sponge DNA sample includes a COl (mitochondrial cytochrome c oxidase subunit Ⅰ) gene region of a sponge mitochondrial DNA (mtDNA).
The step of obtaining the melting curve according to temperature may include a step of obtaining a melting peak curve for each temperature from the obtained melting curve for each temperature, wherein the step of discriminating the red sea species comprises: It is possible to distinguish species.
In addition, the discrimination of the above-described irrigation species may be performed by discriminating the irrigation species in comparison with the melting temperature of the irrigation type at which the above-mentioned melting temperature is known.
In addition, another embodiment of the present invention is a peptide nucleic acid kit for discriminating hippocampal species, which comprises the above-mentioned peptide nucleic acid set according to the present invention.
Here, the peptide nucleic acid set includes a first group tube and a second group tube containing at least one peptide nucleic acid, and at least one reporter class of the peptide nucleic acid contained in the first group tube may be a peptide nucleic acid This can be the same as one or more reporter types.
Also, the first group tube comprises a peptide nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the second group tube comprises a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: And a peptide nucleic acid, respectively.
The details of other embodiments are included in the detailed description and drawings.
According to the present invention, the melting temperature (Tm) is different for each hongo species using PNA having a strong binding force with DNA, so that the hongae species can be identified easily, quickly, and accurately.
In addition, the peptidic nucleic acid of the present invention may have a structural modification, including a sequence corresponding to a single base polymorphism (SNP) region of the hippocampus in the middle of the base sequence, The difference in melting temperature (Tm) with the target match nucleic acid (perfect match) can be further increased.
In addition, since the peptide nucleic acid for discriminating the mare species according to the present invention exhibits different melting temperatures (Tm) depending on the base sequence (or SNP) to be bound, two or more base sequences are detected with one peptide nucleic acid It is also possible to use two or more peptide nucleic acids in a single channel or tube.
FIG. 1 is a conceptual diagram for explaining technical characteristics of a peptide nucleic acid for discriminating hippocampus according to a preferred embodiment of the present invention,
FIG. 2 is a schematic view for explaining a step of hybridization and a step of obtaining a fusion curve of a method of identifying a hippocampus using a peptide nucleic acid according to another preferred embodiment of the present invention,
FIG. 3 is a graph for explaining an example of a step of obtaining a fusion peak curve from a fusion curve in a method of identifying a hippocampus using a peptide nucleic acid according to the present invention,
FIG. 4 is a schematic view for explaining an example of detecting different melting temperatures (Tm) using a peptide nucleic acid for discriminating mare species according to the present invention,
Figs. 5-11 are illustrative of the present invention, in accordance with the present invention, in accordance with the present invention, wherein Zearaja chilensis, Zearaja maugeana, Raja pulchra, Raja binoculata, Okamejei kenojei, (Dipturus argentinensis), a SNP and a nucleotide sequence of a peptide nucleic acid derived therefrom,
FIG. 12 is a gene position diagram for explaining an example of a base mutation part included in a peptide nucleic acid on the hippocampal COI gene according to the present invention,
13 is a graph for explaining an example of a process of hybridizing a sponge DNA sample and raising the temperature of the hybridized product according to an embodiment of the present invention,
14 to 20 are diagrams showing the peptide nucleic acids of SEQ ID NO: 1 to SEQ ID NO: 6, respectively, according to the present invention, respectively, in the order of Zearaja chilensis, Zearaja maugeana, Raja pulchra, binoculata, Okamejei kenojei, and Dipturus argentinensis, respectively, and the results are shown in FIG.
FIG. 21 is an example of a table summarizing the melting temperature (Tm) and the number of each of the mismatch sequences obtained from the graph of the melting curve for each temperature in FIG. 14 to FIG.
BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprises" or "having" and the like are used to specify that there is a feature, a number, a step, an operation, an element, a component or a combination thereof described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.
The terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.
The 'target nucleic acid' of the present invention means a nucleic acid sequence to be detected, and is annealed or hybridized with a primer or a probe under hybridization, annealing or amplification conditions. The 'target nucleic acid' is not different from the term 'target nucleic acid', 'target nucleic acid sequence' or 'target sequence' as used herein, and is used in this specification.
"Hybridization" of the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur either in perfect match between two nucleic acid strands, or even in the presence of some mismatching bases. The degree of complementarity required for hybridization can vary depending on the hybridization conditions, and can be controlled, in particular, by temperature.
The 'base mutation' of the present invention is characterized by a mutation in the base sequence of the target nucleic acid, which includes not only a single nucleotide polymorphism (SNP) but also a nucleotide substitution, deletion or insertion, , The PNA probe of the present invention can be analyzed by melting curve analysis that the SNP of the target nucleic acid or the base of the target nucleic acid has been substituted, deleted or inserted so that the mutation occurs.
The peptide nucleic acid (PNA) set for identifying a fish species according to the present invention comprises at least one peptide nucleic acid, and the peptide nucleic acid comprises at least one base sequence selected from the group consisting of SEQ ID NOS: 1 to 6 .
That is, the peptide set for peptide discrimination may comprise 1 to 6 PNAs, and the present invention may include a PNA having the nucleotide sequence of SEQ ID NO: 1, a PNA having the nucleotide sequence of SEQ ID NO: 2, A PNA having a nucleotide sequence of SEQ ID NO: 4, a PNA having a nucleotide sequence of SEQ ID NO: 5, and / or a PNA having a nucleotide sequence of SEQ ID NO: 6 .
In general, peptide nucleic acid (PNA) was first synthesized by Nielsen et al. In 1991 as a pseudo-DNA in which a nucleic acid base is linked by a peptide bond rather than a phosphate bond. PNAs are synthesized by artificial chemical methods, not found in nature. PNAs form double strands through hybridization reactions with natural nucleic acids of complementary base sequences. When the length is the same, the PNA / DNA double strand is more stable than the DNA / DNA double strand, and the PNA / RNA double strand is more stable than the DNA / RNA double strand. In addition, PNA is more capable of detecting single nucleotide polymorphism (SNP) than native nucleic acid because the double strand becomes unstable due to single base mismatch. PNA is chemically stable and biologically stable because it is not degraded by nuclease or protease. In addition, PNA is a gene recognition material such as LNA (Locked Nucleic Acid) and MNA (Mopholino nucleic acid), and the basic skeleton is composed of polyamide. PNA has an excellent affinity and selectivity, and has a high thermal / chemical property and stability, which is easy to store and does not easily decompose.
In addition, the PNA-DNA binding power is superior to the DNA-DNA binding force, and the PNA has a melting temperature (Tm) of about 10 to 15 ° C even for one nucleotide miss match. The present invention is to detect nucleotide changes of SNP and InDel using the difference of binding force. Also, since the Tm value varies depending on the nucleotide of the DNA that binds complementarily to the nucleotide of the PNA probe, the present invention uses this to discriminate the species of the sponge. In addition, the PNA probe can be analyzed using a different hybridization methode from the hydrolysis methode of the TaqMan probe. Molecular beacon probes and scorpion probes are similar probes.
For this purpose, the peptide nucleic acid preferably has a reporter and a quencher at both ends thereof. That is, in the PNA probe according to the present invention, a reporter and a quencher capable of quenching the reporter fluorescence may be combined at both ends. The reporter may be selected from the group consisting of 6-carboxyfluorescein, Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein) And the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto. Preferably, Dabcyl is used .
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram for explaining technical characteristics of a peptide nucleic acid for discriminating hippocampus in accordance with a preferred embodiment of the present invention. As shown therein, the PNA probe according to the present invention hybridizes with a target nucleic acid, And rapidly melts from the target nucleic acid at the optimum melting temperature (Tm) of the probe as the temperature rises, thereby extinguishing the fluorescence signal. The present invention is capable of detecting the presence or absence of base mutation of a target nucleic acid through a fluorescence melting curve analysis (FMCA) obtained from the fluorescence signal according to the temperature change at a high resolution. The PNA probe according to the present invention exhibits a predicted melting temperature (Tm) value in the case of perfect match with the target nucleic acid sequence, but incompletely hybridizes with the target nucleic acid in which the base mutation is present, And has a low melting temperature (Tm) value.
The present inventors made comparative analysis of various kinds of hongah genes to prepare PNAs each containing one or more base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, and using this PNA, a melting curve As a result of confirming the melting temperature (Tm) from the above-mentioned melting curve, as can be seen from the examples described later, different results were obtained by the different kinds of the sponges. The present invention uses different PNAs having a strong binding force with DNA to exhibit different melting temperatures (Tm) for different species of ponies, so that the ponies can be identified easily, quickly, and accurately.
Although the length of the PNA nucleotide sequence according to the present invention is not particularly limited, the length of the PNA nucleotide sequence according to the present invention can be 9 to 15 mer lengths including the SNP of the hippocampus. It is also possible to design the probe to have the desired Tm value by adjusting the length of the PNA probe, or to adjust the Tm value by changing the base sequence of the PNA probe of the same length. In addition, since PNA has better binding ability than DNA and has a higher basic Tm value, it can be designed to have a shorter length than DNA, so that it is possible to detect neighbor SNPs. According to the existing HRM method, the difference of the Tm values is very low at about 0.5 ° C., and therefore, additional analysis programs or detailed temperature changes or corrections are required. Therefore, when two or more SNPs appear, analysis is difficult. PNA probes are not affected by probe sequences and SNPs and can be easily analyzed.
In addition, the PNA probe of the present invention is characterized in that, for the difference between the melting temperature (Tm) of the target nucleic acid having the target nucleic acid and the base mutation, the base mutation position of the target nucleic acid is not the center position of the PNA probe, end positionl. < / RTI > If the base corresponding to the end position of the PNA probe is deleted, since the base mutation exists at the end of the probe, it is possible to obtain a perfect match, which is a perfect hybridization, and a fusion of the DNA oligomer having a base mutation There is a disadvantage that analysis is difficult because the difference in temperature (Tm) is small. In contrast, when the base mutated portion is located at the center position of the probe, it makes a structural difference of the probe, and the PNA probe becomes bonded while forming a loop, and due to such a structural difference, Tm).
For this reason, when the PNA probe according to the present invention contains a base sequence in a range of 13 to 17, a sequence corresponding to the single base polymorphism (SNP) region of the hippocampus at one or more of the 6th to 9th positions . Such a PNA may have a structural modification including a sequence corresponding to the SNP region of the horses in the middle of the nucleotide sequence, and thereby, a difference in melting temperature (Tm) with a perfect match, which achieves complete hybridization, Can be greatly increased.
FIG. 2 is a schematic diagram for explaining a step of hybridization and a step of obtaining a fusion curve of a method for identifying a hippocampus species using a peptide nucleic acid according to another preferred embodiment of the present invention.
As shown herein, another embodiment of the present invention is a method of amplifying a sponge DNA sample comprising: (S10) hybridizing a sample of a sponge with a peptide nucleic acid of a peptide nucleic acid set according to the present invention; (S20) of obtaining a temperature-dependent melting curve while increasing the temperature of the hybridized product; And a step (S30) of discriminating the mare species from the melting temperature of the obtained melting curve.
The hybridization step (S10) is to react the PNA according to the present invention with a sponge DNA sample. This step may involve a PCR procedure and it is possible to use a Forward / Reverse primer set for PCR. Such hybridization steps and PCR conditions may include all various methods well known to those of ordinary skill in the art (hereinafter referred to as " those skilled in the art "). It is also possible to include a melting step after the PCR is finished.
Here, it is preferable that the sponge DNA sample includes a COl (mitochondrial cytochrome c oxidase subunit Ⅰ) gene region of a sponge mitochondrial DNA (mtDNA). In general, mtDNA has a faster evolution rate than nuclear DNA (nDNA), and has more mtDNA in one cell, making it more effective for species-specific sequencing than DNA in nuclei with low specificity. In addition, mitochondria are a maternal hereditary genetic recombination that does not occur and has intraspecific and interspecific gradual changes and is useful for population genetic diversity analysis to reveal phylogenetic relationships of taxa. In particular, the mitochondrial cytochrome c oxidase subunit Ⅰ (CO1) gene is useful as a species discrimination marker as a DNA barcode. Although the total length of the CO1 gene is about 1,500 bp, it is also possible to use only about 600 bp which has a sufficient species identification resolution since the nucleotide sequence can be known by one analysis for species discrimination analysis.
In addition, although there are no particular limitations on the kind of the hippocampus capable of discriminating species by the PNA according to the present invention, it is preferable that the species of the hippocampus are Zileaja chilensis, Zearaja maugeana, Raja pulchra, , Raja binoculata, Okamejei kenojei, and Dipturus argentinensis. The term " mollusk species " is not limited to the indicated origin, but includes all species or individuals having a gene sequence identical or similar to the respective mollusk species described above.
Then, the step (S20) of obtaining the temperature-dependent melting curve is for obtaining the melting temperature (Tm) of the sponge DNA sample. For this purpose, fluorescence intensities are measured while increasing the temperature of the hybridized product, and fluorescence intensities according to temperature can be obtained as temperature-dependent melting curves. In other words, FMCA (Fluorescence Melting Curve Analysis) can be used as a method of hybridization methode analysis. The FMCA analyzes the difference in binding force between a product produced after completion of the PCR reaction and a putative probe by Tm. For example, a Tm value can be obtained by measuring the intensity of fluorescence every 1 ° C increase by using a general real-time PCR apparatus.
Next, the step (S30) of discriminating the mare species discriminates the mare species from the melting temperature of the obtained melting curve. For example, the melting temperature of the obtained melting curve can be compared with the known melting temperature of the hippocampus to identify the hippocampus species. The melting curves to which the PNA according to the present invention is applied have different melting temperatures (Tm) (refer to Figs. 14 to 20 and Fig. 21), and the difference of the sphagnum species can be discriminated for each species.
In addition, FIG. 3 is a graph for explaining an example of a step of obtaining a fusion peak curve from a fusion curve in a method of identifying a hippocampus species using a peptide nucleic acid according to the present invention. As shown in the figure, the slope value of the fusion curve is used , And the melting peak curves are obtained for each temperature. The melting peak curves are easy to grasp the melting temperature (Tm) of the sphagnum species. The step (S20) of obtaining the melting curve according to temperature includes a step (S21) of obtaining a melting peak curve according to temperature from the obtained melting curve according to temperature, and the step (S30) It is possible to distinguish the sperm species from the melting temperature of the obtained melting peak curve.
On the other hand, another embodiment of the present invention is a peptide nucleic acid kit for discriminating hippocampal species, which comprises the above-mentioned peptide nucleic acid set according to the present invention.
The kit of the present invention can optionally include reagents necessary for conducting a target amplification PCR reaction (e. G., PCR reaction) such as a buffer, a DNA polymerase joiner and deoxyribonucleotide-5-triphosphate. In addition, the kit of the present invention may also comprise various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
Using such a kit, it is possible to effectively detect a single base mutation of a target nucleic acid and a mutation due to a deletion or insertion of a base through analysis of a dissolution curve by a PNA probe, thereby enabling identification of the species of the skunk.
Here, another feature of the present invention resides in that the peptide nucleic acid set includes a first group tube and a second group tube each containing at least one peptide nucleic acid, and at least one kind of a reporter (fluorescent substance) possessed by the peptide nucleic acid contained in the first group tube May be the same as one or more types of reporters of the peptide nucleic acid contained in the second group tube. That is, although the PNA included in the first group tube has different reporters and has the same kind of reporter as the PNA included in the second group tube, the first group tube and the second group tube are separated from each other, The number of reporters to be used can be minimized.
4 is a schematic diagram for explaining an example of detecting different melting temperatures (Tm) using a peptide nucleic acid for discriminating mare species according to the present invention. As shown in the figure, the PNA Probes are also relatively easy to analyze in heterozygous samples with two melting curve graphs, and two or more nucleotide variations adjacent to each other can be analyzed simultaneously.
In the present invention, the first group tube comprises a peptide nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the second group tube comprises SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: The nucleotide sequence of SEQ ID NO: 5 (SSRaCP24) and the nucleotide sequence of SEQ ID NO: 2 (SSRaCP19) are located very close to each other, as shown in the following Examples. (See Fig. 12), it is advantageous to exclude them from the same combination.
The present invention may be better understood by the following examples, which are for the purpose of illustrating the invention and are not intended to limit the scope of protection defined by the appended claims.
Example
1: Discrimination
Peptide hexane
Produce
Among the various species of red flags in this embodiment, there are a variety of red sea bream species, including Zearaja chilensis, Zearaja maugeana, Raja pulchra, Raja binoculata, Okamejei kenojei, Dipturus argentinensis) were selected.
Then, a COI primer was prepared to perform PCR using the DNA extracted from the skeletal tissue as a template.
(SEQ ID NO: 9), Z. maugeana (SEQ ID NO: 10), R. pulchra (SEQ ID NOs: 7 and 8), R. binoculata . The nucleotide sequences of the mitochondrial COI genes of six species of the rice species of argentinensis (SEQ ID NO: 11) were analyzed, and SNPs showing the significance of each species were found by comparing the nucleotide sequences. Based on the nucleotide sequences, specific nucleotide sequences were selected .
FIGS. 5 to 11 are sequence diagrams showing a part of the COI gene, the SNP and the base sequence of PNA derived from the part of the COI gene of the six hippocampus according to the present invention, Is a gene position diagram for explaining an example of the base mutation part included. 5 to 11, the SNPs of the respective species are represented by red bolds, and the base sequence of the PNA according to the present invention based on the SNPs is indicated in bold in a yellow box. In FIG. 12, each bp represents a position corresponding to the first base of the PNA according to the present invention. The number of each nucleotide sequence was determined on the basis of R. pulchra , and the PNA probe was derived to give a perfect match, and the base sequence of PNA according to the present invention was derived.
For example, the O. kenojei species has "C" as the SNP at position 117, and thus the nucleotide sequence corresponding to position 110 to 123 (SEQ ID NO: 4) centering on position 117 is used in the present invention , And this probe (SSRaCP05) is suitable for discriminating O. kenojei species.
In addition, Z. chilensis, Z. maugeana, O. kenojei, and D. argentinensis at positions 343 have different bases from R. pulchra and R. binoculata, and based on this, positions corresponding to positions 339 to 351 The nucleotide sequence (SEQ ID NO: 6) was composed of the base sequence of PNA according to the present invention, and these probes (SSRaCP11) were Z. chilensis, Z. maugeana, O. kenojei, and D. argentinensis, R. pulchra and R. binoculata . ≪ / RTI >
Thus, the nucleotide sequence of PNA according to the present invention was determined and shown in Table 1 below.
In Table 1, O means linker and K means lysine.
The combination of PNA probes was SSRaCP22, SSRaCP19 and SSRaCP08 as the first group, and SSRaCP23, SSRaCP24 and SSRaCP11 as the second group. Each combination was made so that the same fluorescence was not included. SSRaCP24 and SSRaCP19 are excluded from the same combination because they are located very close to each other as shown in FIG.
Then, a PNA probe according to the present invention was prepared using the nucleotide sequence shown in Table 1, the reporter and the quencher. PNA probes were designed using a PNA probe designer (Applied Biosystems, USA). All PNA probes (FAM-labeled, Dabcyl) used in the present invention were synthesized by HPLC purification method in PANAGENE, Korea. The purity of all synthesized probes was confirmed by mass spectrometry, The unnecessary secondary structure of the probe was avoided for effective coupling.
Example
2: 6 kinds of skates
DNA
Dissolution curve analysis for sample
Using the PNA according to the present invention fabricated according to Example 1, a dissolution curve was determined for six samples of the hongah language DNA, and the analysis was performed to determine the species.
PCR was performed using a CFX96 ™ Real-Time system (BIO-RAD, USA). Asymmetric PCR was used to generate a single-stranded target nucleic acid under all experimental conditions. The conditions of asymmetric PCR are as follows; 1X Bio-eye in real time so that the
FIG. 13 is a graph for explaining an example of a process of hybridizing a hoe's DNA sample and raising the temperature of the hybridized product according to an embodiment of the present invention. As shown here, the real-time PCR process was denatured at 95 ° C for 10 minutes, then reacted at 95 ° C for 30 seconds, 50 ° C for 30 seconds, and 74 ° C for 45 seconds, Lt; RTI ID = 0.0 > 1 C < / RTI > at the end of one cycle. This is to ensure that the COI gene of the skull is amplified accurately. 50 cycles were repeated and fluorescence was measured in real time. Melting curve analysis was performed by denaturation at 95 ° C for 3 minutes, followed by hybridization at 35 ° C for 5 minutes, followed by a melting curve analysis in which fluorescence was measured by increasing the temperature from 20 ° C to 80 ° C by 1 ° C. The stationary state was maintained for 10 seconds between each step (see Fig. 13).
14 to 20 are diagrams showing the peptide nucleic acids of SEQ ID NO: 1 to SEQ ID NO: 6, respectively, according to the present invention, respectively, in the order of Zearaja chilensis, Zearaja maugeana, Raja pulchra, binoculata, Okamejei kenojei, and Dipturus argentinensis, respectively, as shown in Fig. In each of FIGS. 14 to 20, the six melting curves were carried out in one tube in the above three, and three in the bottom three tubes. The three above are SSRaCP22 (FAM), SSRaCP08 (Texas Red), SSRaCP19 (Cy5) from the left and SSRaCP23 (FEX), SSRaCP11 (HEX) and SSRaCP24 (Texas Red) from the left. The number of graph lines on each melting curve is a measure of the different samples of the species.
As shown in FIG. 14 to FIG. 20, it can be confirmed that the melting curves to which the PNA according to the present invention is applied have different melting temperatures (Tm). In addition, it can be confirmed that the difference in melting temperature (Tm) occurs between the hippocampus having the SNP at a specific position and the true hippocampus (R. pulchra 1, perfect match) showing complete hybridization.
FIG. 21 is an example of a table summarizing the melting temperature (Tm) and the number of each of the mismatch sequences obtained from the graph of the melting curve for each temperature in FIG. 14 to FIG. In the table of FIG. 21, 0 indicates a perfect match due to the number of nucleotides of the miss match, and 1 through 3 indicates the number of each miss match nucleotide. Also, the blue part indicates the perfect match.
Accordingly, it can be seen that at least one perfect match appears in all species when the PNA according to the present invention is used. This is in agreement with the intention of constructing the base sequence of PNA according to the present invention. Through this, it is possible to distinguish the spermatozoa using the PNA according to the present invention.
For example, in FIG. 21, SSRaCP05 has two miss matches only for O. kenojei species, and Tm is also distinct from other species. Also in FIG. 21, SSRaCP11 has one miss match for Z. chilensis, Z. maugeana, and D. argentinensis species, and Tm is also different from R. pulchra and R. binoculata. In case of O. kenojei, the fusion temperature is not shown by SSRaCP11, which is not hybridized or melted due to three miss matches.
Example
3: Identification method of the mollusks according to the score by melting temperature
In the case of using the PNA according to the present invention to discriminate a species from an unknown sponge DNA sample, it is possible to make a score table for each melting temperature in advance and utilize it.
Specifically, the melting curve analysis was performed as in Example 2, and the obtained Tm value was digitized according to the perfect match temperature as shown in Table 2 below. That is, a range of ± 3 ° C of the perfect match temperature was made, and when the Tm value of the unknown spike DNA sample was within the above range, a value of 1 was given. Otherwise, a value of 0 was assigned to each sample to have a unique value .
Then, the Tm value obtained in Example 2 is converted into 1 and 0 according to the score in Table 2, and can be expressed as shown in Table 3 below.
Based on the score table as shown in Table 3 above, a Hongyong species discrimination program was produced. It was found that by comparing the Tm value of the unknown spongy DNA sample with respect to the PNA according to the present invention to the score value in Table 3, Can be determined simply, accurately, and quickly.
While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be apparent to those skilled in the art that changes may be made.
Attach an electronic file to a sequence list
Claims (10)
Wherein the peptide nucleic acid comprises at least one base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, respectively.
Wherein the peptide nucleic acid has a reporter and a quencher at both ends of the peptide nucleic acid.
Wherein each of the one or more base sequences comprises a base in a range of 13 to 17 and has a sequence corresponding to a single base polymorphism (SNP) region of the hippocampus at one or more of the 6th to 9th positions. Set of peptide nucleic acid for species discrimination.
Obtaining a temperature-dependent melting curve while increasing the temperature of the hybridized product; And
And determining a mallard species from the melting temperature of the obtained melting curve.
Wherein the sponge DNA sample comprises a COl (mitochondrial cytochrome c oxidase subunit I) gene region of a sponge mitochondrial DNA (mtDNA).
The step of obtaining the temperature-dependent melting curve includes the step of obtaining a temperature-dependent melting peak curve from the obtained temperature-dependent melting curve,
Wherein the step of discriminating the mare species discriminates the mare species from the melting temperature of the obtained melting peak curve.
Wherein the determination of the mare species is made by comparing the melting temperature of the mare species with the known melting temperature of the mare species to identify the mare species.
Wherein said set of peptide nucleic acids comprises a first group tube and a second group tube comprising at least one peptide nucleic acid,
Wherein at least one reporter type of the peptide nucleic acid contained in the first group tube is the same as at least one reporter class of the peptide nucleic acid contained in the second group tube.
Wherein said first group tube comprises a peptide nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3,
Wherein the second group tube comprises a peptide nucleic acid comprising a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101720483B1 (en) | 2016-01-11 | 2017-03-28 | 대한민국 | Peptide nucleic acids set for identifying Anguilliformes species and identifying method of Anguilliformes species using the same |
| KR101775219B1 (en) | 2015-06-24 | 2017-09-06 | 배재대학교 산학협력단 | Probe for identifying sea bream species and tilapia species and identifying method using the same |
| KR101817107B1 (en) | 2015-09-02 | 2018-01-10 | 대한민국 | Method For Identification Of Beringraja pulchra Using Real-Time PCR assay |
| KR101940977B1 (en) | 2017-09-13 | 2019-01-23 | 대한민국 | Primer set for identifying skate ray, imported skate ray and stingray, analysis kit using its primers, and identifying method using thereof |
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| KR100806208B1 (en) * | 2006-08-04 | 2008-02-22 | (주)지노첵 | A Polynucleotide probe and chip kit for skate and ray species discrimination based on the single nucleotide polymorphisms in the mitochondrial COI gene |
| KR20090066972A (en) * | 2007-12-20 | 2009-06-24 | 한국생명공학연구원 | Probe and dna chip for identifying avian species |
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| KR100806208B1 (en) * | 2006-08-04 | 2008-02-22 | (주)지노첵 | A Polynucleotide probe and chip kit for skate and ray species discrimination based on the single nucleotide polymorphisms in the mitochondrial COI gene |
| KR20090066972A (en) * | 2007-12-20 | 2009-06-24 | 한국생명공학연구원 | Probe and dna chip for identifying avian species |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101775219B1 (en) | 2015-06-24 | 2017-09-06 | 배재대학교 산학협력단 | Probe for identifying sea bream species and tilapia species and identifying method using the same |
| KR101817107B1 (en) | 2015-09-02 | 2018-01-10 | 대한민국 | Method For Identification Of Beringraja pulchra Using Real-Time PCR assay |
| KR101720483B1 (en) | 2016-01-11 | 2017-03-28 | 대한민국 | Peptide nucleic acids set for identifying Anguilliformes species and identifying method of Anguilliformes species using the same |
| KR101940977B1 (en) | 2017-09-13 | 2019-01-23 | 대한민국 | Primer set for identifying skate ray, imported skate ray and stingray, analysis kit using its primers, and identifying method using thereof |
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